Fatty acid activation of the reconstituted brown adipose tissue mitochondria uncoupling protein

The effect of fatty acids, palmitoyl-CoA, and N',N-dicyclohexylcarbodiimide on the ion conductance of the reconstituted brown adipose tissue mitochondria uncoupling protein was investigated. 1, 5, and 10 microM palmitic acid induced a specific, GDP inhibited, increase in proton conductance in proteoliposomes containing the uncoupling protein but not in proteoliposomes prepared with purified protein extracts of liver mitochondria. 10 microM oleic acid, like palmitic acid, increased proton conductance in proteoliposomes prepared with the uncoupling protein. Palmitoyl-CoA and caprylic acid had no effect on increasing proton conductance. Similar to the observation in mitochondria, there was no effect of palmitic acid on Cl-conductance, but unlike mitochondria its activation by palmitoyl-CoA or inhibition by N',N-dicyclohexylcarbodiimide was lost. The results, obtained in an isolated system, provide support for the contention that long chain fatty acids act as an acute physiological activator of the uncoupling protein.     

Uncoupling activities of chalcones and dihydrochalcones on isolated mitochondria from potato tubers and mung bean hypocotyls

The uncoupling properties of 23 chalcones and dihydrochalcones were studied. Twelve compounds completely uncouple oxidative phosphorylation in mung bean hypocotyl and potato tuber mitochondria, four are weak uncouplers and seven are without effect. Usually, mung bean mitochondria are more sensitive to uncoupling agents than potato mitochondria. The uncoupling activity of chalcones and dihydrochalcones appears to be connected with the presence of hydrogen or hydroxyl groups in the 2′-position and hydrogen, hydroxyl or nitrate groups in the 4′-position. The α-β-unsaturated carbonyl system is not essential for activity. For the compounds which are not very lipophilic, substituents on the B-ring are without effect on the uncoupling properties. Phloretin appears to be an active uncoupler; its 6′-glucoside is without effect.     

Assessment of uncoupling activity of uncoupling protein 3 using a yeast heterologous expression system.

Uncoupling protein 3L, uncoupling protein 1 and the mitochondrial oxoglutarate carrier were expressed in Saccharomyces cerevisae. Effects on different parameters related to the energy expenditure were studied. Both uncoupling protein 3L and uncoupling protein 1 reduced the growth rate by 49% and 32% and increased the whole yeast O2 consumption by 31% and 19%, respectively. In isolated mitochondria, uncoupling protein 1 increased the state 4 respiration by 1.8-fold, while uncoupling protein 3L increased the state 4 respiration by 1.2-fold. Interestingly, mutant uncoupling protein 1 carrying the H145Q and H147N mutations, previously shown to markedly decrease the H+ transport activity of uncoupling protein 1 when assessed using a proteoliposome system (Bienengraeber et al. (1998) Biochem. 37, 3-8), uncoupled the mitochondrial respiration to almost the same degree as wild-type uncoupling protein 1. Thus, absence of this histidine pair in uncoupling protein 2 and uncoupling protein 3 does not by itself rule out the possibility that these carriers have an uncoupling function. The oxoglutarate carrier had no effect on any of the studied parameters. In summary, a discordance exists between the magnitude of effects of uncoupling protein 3L and uncoupling protein 1 in whole yeast versus isolated mitochondria, with uncoupling protein 3L having greater effects in whole yeast and a smaller effect on the state 4 respiration in isolated mitochondria. These findings suggest that uncoupling protein 3L, like uncoupling protein 1, has an uncoupling activity. However, the mechanism of action and/or regulation of the activity of uncoupling protein 3L is likely to be different.     

Uncoupling effect of anacardic acids from cashew nut shell oil on oxidative phosphorylation of rat liver mitochondria

Anacardic acids are one of natural products found in not only the cashew nut shell oil but also the nut and fruit juice. The present study was conducted to investigate the uncoupling effect of anacardic acids on oxidative phosphorylation of rat liver mitochondria using succinate (plus rotenone) as a substrate. Four anacardic acids with C15:0, C15:1, C15:2 or C15:3 as an alkyl side chain exhibited uncoupling effects similar to the classical uncoupler, 2,4-dinitrophenol on ADP/O ratio, state 4 and respiratory control ratio (RCR). Anacardic acid with C15:1 side chain was most effective for uncoupling of these compounds. Salicylic acid, which has no alkyl side chain, exhibited a very weak uncoupling effect on oxidative phosphorylation. When the carboxyl group in anacardic acids was lost converting them to the corresponding cardanols, uncoupling activity dramatically decreased regardless of the number of double bonds in the long alkyl chain. These results suggest that the C15 alkyl side chain as well as the carboxyl group may play an important role in assisting the uncoupling activity of anacardic acids in liver mitochondria of animals. This study provides the first evidence of an uncoupling effect of anacardic acids on liver mitochondria     

Free radicals alter maximal diaphragmatic mitochondrial oxygen consumption in endotoxin-induced sepsis

Recent studies indicate that sepsis is associated with enhanced generation of several free radical species (nitric oxide, superoxide, hydrogen peroxide) in skeletal muscle. While studies suggest that free radical generation causes uncoupling of oxidative phosphorylation in sepsis, no previous report has examined the role of free radicals in modulating skeletal muscle oxygen consumption during State 3 respiration or inhibiting the electron transport chain in sepsis. The purpose of the present study was to examine the effects of endotoxin-induced sepsis on State 3 diaphragm mitochondrial oxygen utilization and to determine if inhibitors/scavengers of various free radical species would protect against these effects. We also examined mitochondrial protein electrophoretic patterns to determine if observed endotoxin-related physiological derangements were accompanied by overt alterations in protein composition. Studies were performed on: (a) control animals, (b) endotoxin-treated animals, (c) animals given endotoxin plus PEG-SOD, a superoxide scavenger, (d) animals given endotoxin plus L-NAME, a nitric oxide synthase inhibitor, (e) animals given only PEG-SOD or L-NAME, (f) animals given endotoxin plus D-NAME, and (g) animals given endotoxin plus denatured PEG-SOD. We found: (a) no alteration in maximal State 3 mitochondrial oxygen consumption rate at 24 h after endotoxin administration, but (b) a significant reduction in oxygen consumption rate at 48 h after endotoxin, (c) no effect of endotoxin to induce uncoupling of oxidative phosphorylation, (d) either PEG-SOD or L-NAME (but neither denatured PEG-SOD nor D-NAME) prevented endotoxin-mediated reductions in State 3 respiration rates, (e) some mitochondrial proteins underwent tyrosine nitrosylation at 24 h after endotoxin administration, and (f) SDS-page electrophoresis of mitochondria from endotoxin-treated animals revealed a selective depletion of several proteins at 48 h after endotoxin administration (but not at 24 h); (g) administration of L-NAME or PEG-SOD prevented this protein depletion. These data provide the first evidence that endotoxin-induced reductions in State 3 mitochondrial oxygen consumption are free radical-mediated.     

Involvement of phosphate carrier as a part of complex with ADP/ATP and aspartate/glutamate antiporters in palmitic acid-induced uncoupling in liver mitochondria

In liver mitochondria, the phosphate carrier is involved in protonophoric uncoupling effect of fatty acids together with ADP/ATP and aspartate/glutamate antiporters (Samartsev et al. 2003. Biochemistry (Moscow). 68, 618–629). Liver mitochondria depleted of endogenous oxidation substrates (exhausted mitochondria) have been used in the present work. In these mitochondria, like in the intact liver mitochondria, the specific inhibitor of ADP/ATP antiporter (carboxyatractylate) and the substrate of aspartate/glutamate antiporter (aspartate) suppress the uncoupling activity of palmitic acid. It is shown that in exhausted mitochondria the substrate of phosphate carrier (inorganic phosphate) and its nonspecific inhibitor mersalyl partially suppress palmitic acid-induced uncoupling due to decrease in the component of uncoupling activity sensitive to carboxyatractylate and aspartate. In the presence of inorganic phosphate or mersalyl, carboxyatractylate and aspartate added separately subsequent to palmitic acid do not suppress its uncoupling activity. They are effective only when added jointly. In the presence of thiourea or pyruvate, such effects of inorganic phosphate and mersalyl are not observed. It is supposed that in the presence of inorganic phosphate or mersalyl and under the condition of oxidation of critical SH-groups in mitochondria, the phosphate carrier, ADP/ATP antiporter, and aspartate/glutamate antiporter are involved in uncoupling function together with the general fatty acid pool as an uncoupling complex. The role of phosphate carrier in this complex may consist in facilitation of lateral transfer of the fatty acid molecules from one antiporter to another.     

Crystal violet as an uncoupler of oxidative phosphorylation in rat liver mitochondria

Crystal violet exhibited characteristics of an uncoupler of oxidative phosphorylation, i.e. it released respiratory control, hindered ATP synthesis, enhanced ATPase activity, and produced swelling of isolated rat liver mitochondria. Maximal stimulation of respiration, ATPase activity, and swelling was observed at a concentration of 40 microM. The inhibition of State 3 respiration by oligomycin was released by crystal violet. High concentrations of crystal violet inhibited mitochondrial respiration. The uncoupling effect of crystal violet required inorganic phosphate and was abolished by N-ethylmaleimide. The adenine nucleotides ADP and ATP protected mitochondria from uncoupling by the dye. The dye taken up by mitochondria was released into the incubation medium on induction of uncoupling. In the absence of phosphate, the dye did not cause uncoupling, but its retention was much greater than in the presence of phosphate. Crystal violet is suggested to induce uncoupling by acting on the membrane, rather than by its electrophoretic transfer into the mitochondria.     

Toxicity of emestrin,a new macrocyclic dithiodioxopiperazine mycotoxin,to mitochondrial function

The effect of emestrin, a new macrocyclic epidithiodioxopiperazine mycotoxin from severalEmericella species, on mitochondrial reactions was studied using isolated rat liver mitochondria to gain insight into the molecular mechanism for itsin vivo toxicity to rat and mouse. Emestrin was found to inhibit ATP synthesis in mitochondria causing an uncoupling of oxidative phosphorylation and a depression of respiration in isolated mitochondria. In addition to these effects on mitochondrial respiration, emestrin elicited a dratsic structural alteration (swelling) of mitochondria as observed in thein vivo system. The mitochondrial swelling was significantly enhanced by the subsequent addition of calcium ion. Emestrin B, in which dithio group is replaced by trithio group, exerted an uncoupling effect on oxidative phosphorylation without accompanying such depressive effect on state 3 respiration as observed for emestrin.     

Uncoupling of oxidative phosphorylation by divalent cationic cyanine dye. Participation of phosphate transporter

The trinuclear cationic cyanine dye tri-S-C4(5) was found to be an uncoupler of oxidative phosphorylation. Its uncoupling required inorganic phosphate (Pi) or arsenate, which is transported into mitochondria via the Pi transport system, and was abolished by the Pi-transport inhibitor N-ethylmaleimide or mersalyl. The dye stimulated Pi uptake into mitochondria, and its uncoupling action was accompanied by swelling of the mitochondria. The adenine nucleotides ADP and ATP protected mitochondria from uncoupling by the dye. The dye taken up by mitochondria was released into the incubation medium on induction of uncoupling. In the absence of Pi, the dye did not cause uncoupling, but its uptake was much greater than in the presence of Pi. The cyanine dye is suggested to induce uncoupling by acting on the membrane, rather than after its electrophoretic transfer into the mitochondria.     

Effect of quinocitrinines from the fungus Penicillium citrinum on the respiration of yeasts and bacteria

We studied the effect of quinocitrinines on the respiratory activity of yeasts (Yarrowia lipolytica) and bacteria (Arthrobacter globiformis). Quinocitrinines were shown to activate respiration of native cells in both types of organisms. Studies of yeast mitochondria showed that quinocitrinine exerts an uncoupling effect on oxidative phosphorylation, which activates the respiration, reduces the respiratory control, and decreases the ADP/O ratio. Experiments with intact mitochondria and native cells of Arthrobacter globiformis revealed that quinocitrinine decreases the membrane potential. The uncoupling effect likely constitutes a mechanism of the antibiotic activity of quinocitrinines.     

The role of free fatty acids in mitochondrial energetic metabolism in winter wheat seedlings

The effect of fatty acids (FAs) (C12–C24) on the functioning of winter wheat (Triticum aestivum L.) mitochondria was studied. Such fatty acids as C12:0, C16:0, and C18:0 and unsaturated FAs, such as C18:1 (n-9 cis), C18:1 (n-12 cis), C18:2 (n-9, 12), (18:3, n-3), and C22:1 (n-9 cis) caused efficient uncoupling of oxidative phosphorylation in mitochondria, i.e., an increase in the nonphosphorylating respiration rate and a decrease in the respiratory control value. It was established that C16:0 had the strongest uncoupling effect among all saturated FAs, and C18:3, among unsaturated FAs. The uncoupling effect of saturated FAs is provided by the ADP/ATP-antiporter, while plant uncoupling proteins play an important role in the uncoupling effect of unsaturated FAs. In addition, unsaturated, as well as saturated FAs might serve as oxidative substrates for mitochondria. It was concluded that the role of FAs in energetic metabolism of winter wheat seedlings consisted of uncoupling of oxidative phosphorylation and of serving as substrates for oxidation.     

Structural changes in mitochondria induced by uncoupling reagents. The response to snake-venom phospholipase A

Interaction with uncoupling reagents at minimally effective concentrations enhanced the response of freshly isolated rat-liver mitochondria to the action of snake-venom phospholipases. This study corroborated previous findings with proteinases that uncoupling reagents increase the susceptibility of freshly isolated mitochondria to enzymatic attack, as determined by turbidimetric and titrimetric techniques. It is postulated that this effect of uncoupling reagents is due to their binding to protein constituents of the mitochondrial membranes, and a subsequent derangement of the protein-phospholipid organization. The possible relevance of the phenomenon to uncoupling of oxidative phosphorylation is discussed.     

Superoxide stimulates a proton leak in potato mitochondria that is related to the activity of uncoupling protein

The ability of plant mitochondrial uncoupling proteins to catalyze a significant proton conductance in situ is controversial. We have re-examined conditions that lead to uncoupling of mitochondria isolated from the tubers of potato (Solanum tuberosum). Specifically, we have investigated the effect of superoxide. In the absence of superoxide, linoleic acid stimulated a proton leak in mitochondria respiring NADH that was insensitive to GTP. However, when exogenous superoxide was generated by the addition of xanthine and xanthine oxidase, there was an additional linoleic acid-stimulated proton leak that was specifically inhibited by GTP. Under these conditions of assay (NADH as a respiratory substrate, in the presence of linoleic acid and xanthine/xanthine oxidase) there was a higher rate of proton conductance in mitochondria from transgenic potato tubers overexpressing the StUCP gene than those from wild type. The increased proton leak in the transgenic mitochondria was completely abolished by the addition of GTP. This suggests that superoxide and linoleic acid stimulate a proton leak in potato mitochondria that is related to the activity of uncoupling protein. Furthermore, it demonstrates that changes in the amount of StUCP can alter the rate of proton conductance of potato mitochondria.     

Divalent cation chelators citrate and EDTA unmask an intrinsic uncoupling pathway in isolated mitochondria

We demonstrate a suppression of ROS production and uncoupling of mitochondria by exogenous citrate in Mg²⁺ free medium. Exogenous citrate suppressed H₂O₂ emission and depolarized mitochondria. The depolarization was paralleled by the stimulation of respiration of mitochondria. The uncoupling action of citrate was independent of the presence of sodium, potassium, or chlorine ions, and it was not mediated by the changes in permeability of the inner mitochondrial membrane to solutes. The citrate transporter was not involved in the citrate effect. Inhibitory analysis data indicated that several well described mitochondria carriers and channels (ATPase, IMAC, ADP/ATP translocase, mPTP, mKATP) were not involved in citrate’s effect. Exogenous MgCl₂ strongly inhibited citrate-induced depolarization. The uncoupling effect of citrate was demonstrated in rat brain, mouse brain, mouse liver, and human melanoma cells mitochondria. We interpreted the data as an evidence to the existence of a hitherto undescribed putative inner mitochondrial membrane channel that is regulated by extramitochondrial Mg²⁺ or other divalent cations.     

Features of the Uncoupling Effect of Fatty Acids in Liver Mitochondria of Mammals with Different Body Weight

In the presence of oligomycin, EGTA, and magnesium ions, the protonophore uncoupling activity of palmitate (V(Pal)) is determined as the ratio of the acceleration of respiration with palmitate to its concentration. Under these conditions, V(Pal) in liver mitochondria of one-month-old rats with the body weight of 50 g is 1.46-fold higher than in liver mitochondria of adult rats with the body weight of 250 g, whereas the uncoupling activity of FCCP does not depend on the age of the animals. The difference in V(Pal) is mainly due to its component insensitive to carboxyatractylate and glutamate (V(Ins)). This value is 2.9-fold higher in mitochondria of one-month-old rats than in those of adult rats. The protonophore activity of palmitate is similar in liver mitochondria of four-day-old and adult rats. In liver mitochondria of adult mammals (mouse, rat, guinea pig, rabbit), V(Pal) decreases with increase in the body weight of the animals. In double logarithmic coordinates, the dependence of the V(Pal) value on the body weight is linear with slope angle tangent of -0.18. The V(Pal) value is mainly contributed by its component V(Ins). In the presence of calcium ions, palmitate induces the nonspecific permeability of the inner membrane of liver mitochondria (pore opening). This Ca2+-dependent uncoupling effect of palmitate is less pronounced in mitochondria of one-month-old rats than in those of adult rats. In mitochondria of adult animals (mice, rats, and guinea pigs), the Ca2+-dependent uncoupling activity of palmitate is virtually the same. It is concluded that the protonophore uncoupling effect of palmitate in liver mitochondria of mammals, unlike its Ca2+-dependent effect, is associated with thermogenesis at rest and also with production of additional heat on cooling of the animals.     

Comparative study on uncoupling effects of laurate and lauryl sulfate on rat liver and skeletal muscle mitochondria

Uncoupling effects of laurate and lauryl sulfate have been studied in the isolated rat liver and skeletal muscle mitochondria. In the oligomycin-treated liver mitochondria, 0.02 mM laurate or 0.16 mM lauryl sulfate caused a two-fold stimulation of respiration, accompanied by a membrane potential decrease. Carboxyatractylate (CAtr) and glutamate (or aspartate) strongly decrease the effect of laurate and lauryl sulfate on respiratory rate and membrane potential (the recoupling effect). With both uncouplers, this effect is maximal for CAtr and glutamate (aspartate) at pH 7.8 and 7.0, respectively. Tetraphenyl phosphonium cations, which decrease negative membrane charges, cause an alkaline shift of these pH dependences. Small amounts of lauryl sulfate, which increase the membrane negative charge, induce the opposite shift when laurate is used as an uncoupler. ADP, but not GDP, partially recouple with both laurate and lauryl sulfate. We conclude that lauryl sulfate-induced uncoupling in rat liver, like the uncoupling induced by laurate, is mediated by the ATP/ ADP and glutamate/aspartate antiporters. In skeletal muscle mitochondria uncoupled by laurate, 200 microM GDP causes partial recoupling which can be enhanced by a subsequent additions of CAtr, glutamate and serum albumin. CAtr added before GDP promotes a larger recoupling than when added after GDP and prevents the subsequent effect of GDP. ADP is effective as recoupler at lower concentrations that GDP, whereas CDP is without influence. Lauryl sulfate uncoupling of skeletal muscle mitochondria is GDP-resistant but is sensitive to ADP, CAtr, glutamate and serum albumin. Our data suggest that in skeletal muscle mitochondria a GDP-sensitive mechanism is involved in uncoupling induced by laurate. This mechanism is absent in liver mitochondria. Possible mechanisms of laurate and lauryl sulfate-induced uncoupling are discussed.     

Uncoupling effect of fatty acids on heart muscle mitochondria and submitochondrial particles.

The effect of ATP/ADP-antiporter inhibitors on palmitate-induced uncoupling was studied in heart muscle mitochondria and inside-out submitochondrial particles. In both systems palmitate is found to decrease the respiration-generated membrane potential. In mitochondria, this effect is specifically abolished by carboxyatractylate (CAtr) a non-penetrating inhibitor of antiporter. In submitochondrial particles, CAtr does not abolish the palmitate-induced potential decrease. At the same time, bongkrekic acid, a penetrating inhibitor of the antiporter, suppresses the palmitate effect on the potential both in mitochondria and particles. Palmitoyl-CoA which is known to inhibit the antiporter in mitochondria as well as in particles decreases the palmitate uncoupling efficiency in both these systems. These data are in agreement with the hypothesis that the ATP/ADP-antiporter is involved in the action of free fatty acids as natural uncouplers of oxidative phosphorylation.     

Cold-Shock 310-kD Protein Uncouples Oxidative Phosphorylation in Plant Mitochondria

We studied the effects of cold-shock 310-kD protein (CSP310) isolated from winter rye seedlings on the energetic activity of plant mitochondria. CSP310 was shown to enhance nonphosphorylating respiration and uncoupled oxidative phosphorylation in isolated mitochondria. The uncoupling effect was enhanced with increasing protein concentration. An antibody against CSP310 interfered with the uncoupling effect of CSP310. Free fatty acids were not evidently involved in uncoupling. The physiological role of uncoupling between oxidation and phosphorylation during plant adaptation to low temperatures is discussed.     

Redox state of quinone affects sensitivity of Acanthamoeba castellanii mitochondrial uncoupling protein to purine nucleotides

We studied FFA (free fatty acid)-induced uncoupling activity in Acanthamoeba castellanii mitochondria in the non-phosphorylating state. Either succinate or external NADH was used as a respiratory substrate to determine the proton conductance curves and the relationships between respiratory rate and the quinone reduction level. Our determinations of the membranous quinone reduction level in non-phosphorylating mitochondria show that activation of UCP (uncoupling protein) activity leads to a PN (purine nucleotide)-sensitive decrease in the quinone redox state. The gradual decrease in the rate of quinone-reducing pathways (using titration of dehydrogenase activities) progressively leads to a full inhibitory effect of GDP on LA (linoleic acid) induced proton conductance. This inhibition cannot be attributed to changes in the membrane potential. Indeed, the lack of GDP inhibitory effect observed when the decrease in respiratory rate is accompanied by an increase in the quinone reduction level (using titration of the quinol-oxidizing pathway) proves that the inhibition by nucleotides can be revealed only for a low quinone redox state. It must be underlined that, in A. castellanii non-phosphorylating mitochondria, the transition of the inhibitory effect of GDP on LA-induced UCP-mediated uncoupling is observed for the same range of quinone reduction levels (between 50% and 40%) as that observed previously for phosphorylating conditions. This observation, drawn from the two different metabolic states of mitochondria, indicates that quinone could affect UCP activity through sensitivity to PNs.     

Biochemical effects of gossypol in isolated mitochondria: monovalent cations and ATPase activity

1. The effect of gossypol acetic acid upon the energy-producing system of isolated liver mitochondria was studied. Gossypol was found to exert an uncoupling effect. 2. Oxygen consumption and ATPase activity were stimulated only when a monovalent alkali metal cations was present. 3. The mitochondrial proton gradient, produced by the enzymatic hydrolysis of ATP, was lower in the gossypol-treated mitochondria than in the control experiments. 4. It is proposed that gossypol stimulates oxygen consumption and the ATPase activity in mitochondria by a protonophoretic effect and a cation translocation process.